New BODIPYs for photodynamic therapy (PDT): Synthesis and activity on human cancer cell lines

Article abstract of DOI:10.1016/j.bmc.2020.115737

A new class of compounds based on the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene core, known as BODIPYs, has attracted significant attention as photosensitizers suitable for application in photodynamic therapy (PDT), which is a minimally invasive procedur

Full text of DOI:10.1016/j.bmc.2020.115737

Bioorganic & Medicinal Chemistry 28 (2020) 115737
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New BODIPYs for photodynamic therapy (PDT): Synthesis and activity on
human cancer cell lines
a,1
a
a,1,2
a
b
b
Enrico Caruso , Miryam C. Malacarne
, Emanuela Marras , Ester Papa , Linda Bertato ,
a,⁎
Stefano Banfi , Marzia B. Gariboldi
a
b
Department of Biotechnology and Life Sciences (DBSV). University of Insubria, Via J.H. Dunant 3, 21100 Varese (VA), Italy
Department of Theoretical and Applied Sciences (DiSTA). University of Insubria, Via J.H. Dunant 3, 21100 Varese (VA), Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new class of compounds based on the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene core, known as BODIPYs, has
attracted significant attention as photosensitizers suitable for application in photodynamic therapy (PDT), which
is a minimally invasive procedure to treat cancer. In PDT the combination of a photosensitizer (PS), light, and
oxygen leads to a series of photochemical reactions generating reactive oxygen species (ROS) exerting cytotoxic
action on tumor cells.
Photodynamic therapy
BODIPY
Cell death
ROS
Cellular migration
Here we present the synthesis and the study of the in vitro photodynamic effects of two BODIPYs which differ
in the structure of the substituent placed on the meso (or 8) position of the dipyrrolylmethenic nucleus. The two
compounds were tested on three human cancer cell lines of different origin and degree of malignancy.
Our results indicate that the BODIPYs are very effective in reducing the growth/viability of HCT116, SKOV3
and MCF7 cells when irradiated with a green LED source, whereas they are practically devoid of activity in the
dark. Phototoxicity occurs mainly through apoptotic cell death, however necrotic cell death also seems to play a
role. Furthermore, singlet oxygen generation and induction of the increase of reactive oxygen species also appear
to be involved in the photodynamic effect of the BODIPYs. Finally, it is worth noting that the two BODIPYs are
also able to exert anti-migratory activity.
1
. Introduction
overcome the limitations described above and belong to non-porphyrin
photosensitizers, that might be synthetized more easily.
Photodynamic therapy (PDT) is an alternative modality for the
In this respect, over the last decade, a new class of photosensitizers
based on the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene core, com-
treatment of neoplastic and non-malignant lesions in which the com-
bination of a photosensitizing drug (PS), light and oxygen leads to a
series of photochemical reactions generating reactive oxygen species
6
–9
monly known as BODIPYs, has attracted considerable attention.
BODIPYs have been mainly used as fluorescent dyes and for various
1
,2
8,10–12
(
ROS), which are able to cause localized tissue damages.
Despite
other applications.
. The potential use of BODIPYs in PDT has
applications are growing, the use of PDT in clinical oncology is still
limited, mainly due to a number of limitations, such as phototoxic and
photoallergic adverse effects, PSs suboptimal tissue penetration, along
been supported by a number of favourable chemico-physical features
that make these molecules ideal photosensitizers, such as tuneable
hydrophilic/hydrophobic character, high molar extinction coefficients
(generally associate to strong fluorescence), stability under different
environmental conditions, resistance to photobleaching and high light-
3
with problems in obtaining appropriate PSs formulations and dosages.
Furthermore, the most clinically relevant PDT agents are cyclic tetra-
pyrroles (porphyrins, chlorins, and bacteriochlorins) which can require
complex synthetic pathways or difficult chemical modifications to
7
,13–15
dark toxicity ratio.
However, some of these features could also
exert detrimental effects; indeed, if on the one hand photodynamic
activity may benefit from the high chemical stability of the BODIPYs, on
the other hand low biodegradation processes might be associated with
persistent skin photosensitization, by far longer the therapeutic
4
modulate their photophysical and biological properties. Hence, the
synthesis and characterization of new PSs are actively pursued by many
laboratories.3 These new PSs should possess improved properties to
,5
⁎
Corresponding author at: Department of Biotechnology and Life Sciences (DBSV), University of Insubria, Via JH Dunant 3, Varese, Italy.
E-mail address: marzia.gariboldi@uninsubria.it (M.B. Gariboldi).
1
2
The two authors contributed equally to this work.
PhD student of the “Life Science and Biotechnology” course at University of Insubria.
https://doi.org/10.1016/j.bmc.2020.115737
Received 11 June 2020; Received in revised form 6 August 2020; Accepted 22 August 2020
Available online 29 August 2020
0968-0896/ © 2020 Elsevier Ltd. All rights reserved.

E. Caruso, et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115737
necessity. As far as fluorescence is concerned, this property plays a
fundamental role when BODIPYs are exploited as biological probes
while it severely limits their use as photosensitizers, since it indicates
that a large part of the energy absorbed upon excitation is lost through
a process that does not favor the production of cell toxic reactive
oxygen species. Therefore, when a photosensitizing action is required,
high quantum efficiency of fluorescence should be inhibited. To this
purpose, BODIPY structure needs to be modified using substituents with
In order to increase the BODIPYs singlet-to-triplet state crossing,
thereby enhancing singlet oxygen production rate, compounds 3 and 4
were obtained by the insertion of iodine atoms at the 2,6-positions of
compounds 3a and 4a (Scheme 1). This feature is known as “heavy
1
9,22,23
atom effect”.
Regarding the iodination process, we found that the
7
reaction conditions reported in the literature led to low yields of the
desired compound. Therefore, we used milder conditions (treatment
with iodine and iodic acid at 20–25 °C for 24 h) to allow increased
8
,16,17
24
an appropriate oxidation potential.
Several studies focused on
yields (above 55%) in this crucial step.
BODIPY core modifications to increase singlet oxygen generation
showed that such substituents should possess unshared valence electron
pairs to quench the fluorescence of photoexcited BODIPYs, thereby
2.2. Chemico-physical properties
generating relatively long-lived triplet states. Following the interaction
BODIPYs 3 and 4 were isolated as pure compounds as confirmed by
NMR analysis UV–vis absorption spectra and HPLC retention time (Rt).
The comparison of the data obtained for 3 and 4 with those of the non-
iodinated precursors (3a and 4a), allowed an easier determination of
the structure.
3
of these last species with
O , high production of singlet oxygen is
2
7
,18,19
guaranteed.
Another important practical aspect favoring the development of
BODIPYs for PDT comes from their “one-pot” synthetic pathway. As a
matter of fact, the general scheme of BODIPY synthesis involves the
acid-catalyzed reaction, between pyrroles and aldehydes (or acyl
chlorides), yielding dipyrrolylmethane which is first easily oxidized to
dipyrrolylmethene with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
In agreement with results previously published by our group on
other BODIPYs, the iodination process caused a bathochromic shift of
about 30 nm (Table 1), leading to a better overlapping with the emis-
sion profile of the green LED source used in biological and photo-
2
4
(
DDQ) and finally borinated with boron trifluoride (BF
3
· Et
2
O) in the
bleaching experiments. Moreover, the batochromic effect was asso-
ciated with a more than 45% hyperchromic effect, as evidenced by the ε
values. This effect can further improve the PS efficiency: as a matter of
fact, higher absorbances correspond to higher extinction coefficients
(i.e. more energy). In this manner, a high energy absorption should
allow the use of lower PS doses to obtain a significant cell death upon
activation, thus limiting undesirable side effects that may occur when
an excessive PS dosage needs to be used to get an efficient photo-
2
0
presence of a tertiary amine.
In this manuscript we report the synthesis of two BODIPY deriva-
tives (compounds 3 and 4) which differ in the length of the hydro-
carbon chain located in position 4 (para) of the phenyl ring in position 8
(
meso) of the dipyrromethenic core; in particular, compounds 3 and 4
bear a carbon chain of four or eight units respectively, which should
confer different degrees of lipophilicity. This difference should allow to
establish the existence of a correlation between the length of the alkyl
chain and the photodynamic activity of the two BODIPYs. Furthermore,
to make these BODIPY more versatile, allowing for example their at-
tachment by covalent bonds to nanoparticles, each chain features a
2
5
dynamic effect.
Fluorescence data, reported in Table 1, were normalized to the
value obtained using fluorescein as standard. As expected, the values
show that the introduction of two iodine atoms on the BODIPYs 3 and 4
induced a strong decrease in the quantum yield of fluorescence; actu-
ally, the two heavy atoms exerted the known intersystem crossing (ISC)
effect, thus shifting the excited state of singlet to a lower energy triplet
state. This event prompts a nearly complete inhibition of fluorescence
in favor of a very high rate of singlet oxygen generation.
bromine atom in the terminal CH group. This molecule functionali-
2
zation has been previously utilized by our group to improve the in vivo
2
1
bioavailability of porphyrinic derivatives.
The in vitro photodynamic activity of these compounds was assessed
on human cancer cell lines of different origin and degree of malignancy,
namely the colon adenocarcinoma HCT116 cells, the ovarian cancer
SKOV3 cells and the breast adenocarcinoma MCF7 cells, following ir-
radiation with a low energy green LED light, as these molecules are
characterized by intense absorbance in the 495–535 nm range. The
ability of the BODIPYs to induce apoptotic and necrotic response, along
with singlet oxygen and reactive oxygen species production, cellular
uptake, and spontaneous cell migration inhibition, was also evaluated.
As far as LogP values are concerned, it is well known that high
values cannot be determined following spectroscopic analyses of both
octanol and aqueous phases as high-lipophilic compounds do not suf-
2
6
ficiently distribute in the aqueous phase. However, computational
models are available to calculate LogP values from the molecular
2
7,28
structure of the studied compounds.
In this study, LogP values have
been calculated from SMILES (Simplified Molecular Input Line Entry
System) strings, which encode for the molecular structure of the four
BODIPYs listed in Table 2.
2
. Results and discussion
To confirm these empirical data, the evaluation of a relative scale of
lipophilicity of chemical compounds, belonging to a homogeneous
series, has been performed measuring HPLC retention time (Rt) on a
reversed phase (C18) column. Under standardized conditions (i.e. fixed
eluant composition and constant flux), the most lipophilic compound
2.1. Synthesis
The aromatic aldehydes, 4-[(4-bromobutyl)oxy]benzaldehyde (1)
and 4-[(8-bromooctyl)oxy]benzaldehyde (2), used for the synthesis of
the two BODIPYs were obtained by reacting the 4-hydro-
xybenzaldehyde and the appropriate alkyldibromide (1,4-di-
bromobutane or 1,8-dibromooctane), following the procedure reported
2
6
usually shows the highest Rt.
SMILES strings, calculated LogP values and correlations with HPLC
Rt expressed in seconds are reported in Table 2 and Table 3.
2
1
in the literature (Scheme 1).
Results show that, as expected, the ranking obtained in HPLC on the
basis of retention time is the same as the ranking based on lipophilicity
suggested by the calculated LogP (i.e. lipophilicity increases with the
number of atoms and the presence of iodine atoms in the following
order C4 3a < C4 3 < C8 4a < C8 4). These results are further
supported by large correlations reported in Table 3 which show the
consistency of LogP calculations using different computational ap-
proaches and software, compared with HPLC Rt. For this reason, the
average of Crippen LogP and Galas LogP values can be considered as a
fair indicator of the lipophilicity of the four BODIPYs.
The BODIPY derivatives were synthesized by condensation of aro-
matic aldehydes 1 or 2 with 2,4-dimethylpyrrole in the presence of
catalytic amount of trifluoroacetic acid (TFA), following the general
1
5,16
procedure described by Dost and Liu.
The subsequent addition of
2
,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) oxidizes the di-
pyrrolylmethanes to the corresponding dipyrrolylmethenes, which are
then treated with BF ·Et O in the presence of Et N to obtain the desired
3
2
3
BODIPYs. Compounds 3a and 4a were isolated as pure solid after a
single chromatographic step of purification (SiO
ether = 7/3) (Scheme 1).
2
, CH
2
2
Cl /petroleum
Therefore, as mentioned above, BODIPYs with the four carbon
2

E. Caruso, et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115737
Scheme 1. Synthetic approach to obtain the iodinated BODIPYs 3 and 4.
Table 1
Table 3
Wavelength of maximum absorption and extinction coefficient, wavelength of
emission, fluorescent quantum yield and singlet oxygen production for
BODIPYs 3a, 3, 4a and 4.
Correlation matrix among calculated LogP values and HPLC Rt reported in
Table 2.
PADEL Crippen
ACD Galas
HPLC Rt (RP-C18)
1
1
(M-1 cm-
1
2
1O
production3,4
Appendix PS
λ
max
nm)
ε
λ
em
(nm)
Ф
fluo
2
LogP
LogP
seconds
1
(
)
PADEL Crippen LogP
ACD Galas LogP
HPLC Rt (RP-C18)
Seconds
1
C4
C8
3a 503
44,100
66,072
28,200
41,500
542
552
541
554
0.66
0.01
0.52
0.01
n.d.
1.10
n.d.
1.11
0.992
0.997
1
3
533
4a 501
534
0.997
1
4
1
2
DCM was used as solvent.
Φ quantum efficiency of fluorescence with fluorescein (0.1 M NaOH, 0.85)
as reference.
3
Data were normalized vs the decay rate of DPBF in the presence of Rose
Bengal.
4
Determined in isopropanol.
atoms appendix (3a and 3) proved to be less lipophilic than BODIPYs
with the eight carbon atoms appendix (4a and 4). Moreover, the pre-
sence of voluminous and low polar iodine atoms confers a higher li-
pophilicity to iodinated BODIPYs compared to the non-iodinated com-
pounds.
1
The relative rates of singlet oxygen ( O
2
) produced by the BODIPYs
Fig. 1. Photostability of the BODIPYs during irradiation with green LED light.
(
25 μM) were experimentally measured by monitoring the dis-
appearance of the 410 nm absorbance band of the 1,3-diphenyliso-
possibility that different photoactive species could be formed through
the photobleaching process.29
benzofuran (DPBF) in isopropanol and normalized to the value ob-
1
tained using Rose Bengal, a well-known excellent
2
O producer, as
standard. The results, reported in Table 1, indicate that 3 and 4 were
able to produce high levels of singlet oxygen and these levels were
comparable to that produced by Rose Bengal.
2.3. Photodynamic effects in cancer cell lines
The decrease in BODIPYs absorbance intensity during 2 h irradia-
tion with green LED light was evaluated to determine the photostability
percentages of 3 and 4 along with the possible appearance of modified
absorbance profiles, indicating the formation of different species.
The results obtained show that both compounds retained approxi-
mately 40% of the initial absorbance following 2 h irradiation (Fig. 1);
moreover, titration curves indicate that the decay occurred faster
during the first hour than the second hour, suggesting second-order
decay kinetics. No modifications of the absorbance profile were ob-
served for both BODIPY (data not shown). This last result excludes the
The effects of 3 and 4 on HCT116, SKOV3 and MCF7 cell viability,
following 24 h treatment with the BODIPYs, 2 h irradiation with green
LED light and 24 h incubation in drug-free medium in the dark are
reported in Fig. 2, which shows the dose–response curves obtained
according to the MTT assays. The intrinsic cytotoxicity of BODIPYs was
assessed by omitting the irradiation step from the treatment protocol
and it was found negligible in all cases up to concentrations tenfold
higher than those used for PDT experiments (data not shown). Thus, as
a whole these results confirm that irradiation is an essential require-
ment to activate the cytotoxic activity of these compounds.
Table 2
SMILES strings, Crippen LogP and Galas LogP (using PaDEL-Descriptor and ACD/LogP software respectively) and their average (Av. LogP), as well as HPLC Retention
time expressed in seconds for the four BODIPYs (3a, 3, 4a, 4).
Appendix PS SMILES
PADEL Crippen
LogP
ACD Galas
LogP
Av. LogP HPLC Rt (RP-C18)
seconds
C4
C8
3a BrCCCCOc1ccc(cc1)C = 1c2c(C)cc(C)n2[B-](F)(F)[N + ]2 = C(C)C]C(C)C2 = 1
BrCCCCOc1ccc(cc1)C1 = C2C(C) = C(I)C(C) = [N + ]2[B-](F)(F)n2c1c(C)c(I)c2C
4a BrCCCCCCCCOc1ccc(cc1)C = 1c2c(C)cc(C)n2[B-](F)(F)[N + ]2 = C(C)C]C(C)C2 = 1
5.59
6.96
7.15
4.51
5.35
5.51
6.77
5.05
6.15
6.33
7.64
643
856
868
1125
3
4
BrCCCCCCCCOc1ccc(cc1)C1 = C2C(C) = C(I)C(C) = [N + ]2[B-](F)(F)n2c1c(C)c(I)c2C 8.52
3

E. Caruso, et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115737
Fig. 2. Dose-response curves obtained in HCT116, SKOV3 and MCF7 cell lines by the MTT assay, following 24 h treatment with 3 and 4, 2 h irradiation, 24 h
incubation in drug-free medium (mean ± S.E.M. of 3–5 independent experiments).
Table 4
Flow cytometric analysis of BODIPYs-treated cells did not show a
significant arrest in G1 phase, indicating that both the PS does not affect
entry in the cell cycle (Fig. 3) and suggesting that the effect of the
compounds is not generically cytostatic. Cell cycle analysis performed
by omitting the irradiation step were similar to those obtained in
photoactiveted cells (data not shown). Therefore, the two BODIPYs do
not seem to have any significant effect on cell cycle distribution, both in
the presence and in the absence of photoactivation.
IC50 (nM) values obtained in HCT116, SKOV3 and MCF7 cells following
treatment with 3 and 4 for 24 h, 2 h irradiation, 2 h incubation in drug-free
medium and subsequent MTT test. (mean ± S.E.M. of 3–5 independent ex-
periments).
HCT116
SKOV3
MCF7
2.0 ± 0.09 a
3.56 ± 0.28
c
6.88 ± 0.66 e
23.11 ± 0.87
3
4
b
d
11.7 ± 0.84
15.61 ± 1.34
Apoptotic cell death appears to play a central role in tumor cell
a
b
c
p < 0.001 vs MCF7 same compound and p < 0.001 vs 4 same cell line.
p < 0.001 vs MCF7 same compound.
death induced by the two BODIPYs, in agreement to what we and other
30,36–38
authors have described for a number of photosensitizers.
More
p < 0.05 vs MCF7 same compound and p < 0.001 vs 4 same cell line.
p < 0.001 vs MCF7 same compound; e p < 0.001 vs 4 same cell line.
specifically, 24 h exposure to equitoxic concentrations of 3 and 4
(corresponding to the respective IC50 values reported in Table 4), 2 h
irradiation and 24 h incubation in PS-free medium were able to induce
a significant increase in the percentage of apoptotic cells over control in
HCT116, SKOV3 and MCF7 cells (Fig. 4 A). Furthermore, necrotic cell
death seemed to contribute to the photodynamic killing effect of these
BODIPYs in SKOV3 and MCF7 cell lines, as indicate the significant in-
crease of the percentage of necrotic cells observed in these cell lines
following PDT with 3 and 4 (Fig. 4 B), whereas no necrotic cell death
was observed in HCT116 cells under the same experimental conditions.
When the irradiation step was omitted, none of the BODIPYs was
able to trigger an apoptotic or necrotic response in any of the cell lines
(data not shown).
d
The IC50 values reported in Table 4 show that the two BODIPYs
affected HCT116, SKOV3 and MCF7 viability at submicromolar con-
centrations; interestingly, 3 was significantly more potent than 4 in all
cell lines. As already reported for other BODIPYs, differences in potency
can correlate to lipophilicity and to molar extinction coefficient of the
compounds;30] thus, the lower lipophilicity and even the higher molar
extinction coefficient observed for 3, compared to 4 (LogP and ε, re-
spectively), may account for its greater photodynamic activity.
Interestingly, MCF7 cells were significantly more resistant to 3 and
4
than HCT116 and SKOV3 cell lines. Some authors reported that the
final response to PDT could be influenced by both the tumor suppressor
The activation of cell death program in PDT has been reported to
3
1–33
21,30,39
p53 and p53-related proteins.
In particular, wt p53 seems to play
correlate with cellular uptake of photosensitizers.
Therefore, we
an important role in triggering efficient apoptosis in cells following
PDT.31 However, the role of p53 status in the response of cancer cell
lines to PDT is still controversial and conflicting responses to PDT have
performed a spectrophotometric analysis of BODIPYs uptake in
HCT116, SKOV3 and MCF7 cells following 24 h exposition to a high
concentration of 3 and 4 (10 μM). The three cell lines considered dis-
played different extent of intracellular accumulation of the BODIPYs
(Fig. 5); however, only in MCF7 cells the amount of PS which enter the
cells seems to be directly related to the observed higher cytotoxic effect
of 3 with respect to 4.
3
4,35
also been observed in cells with p53 wt.
In agreement with this
disputed situation, HCT116 and MCF7 cells, both expressing wt p53,
showed a very different response to the studied BODIPYs. Maybe the
resistance to 3 and 4 observed in MCF7 cells could be the result of a
different expression of p53-related proteins or other p53 family mem-
bers, but this was not specifically addressed in this work.
In PDT, singlet oxygen and ROS are the effectors of tumor cells
death and light is used to elicit the photosensitizer to induce their
4

E. Caruso, et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115737
Fig. 3. Cell cycle analysis of HCT116, SKOV3 and MCF7 cell lines, following 24 h treatment with equitoxic concentrations of 3 and 4 corresponding to the IC50 values
reported in table IV, 2 h irradiation, 24 h incubation in drug-free medium. Propidium iodide (PI) was used as fluorescent probe (mean ± S.E. of 3 independent
experiments).
production.4
0,41
As reported above, both 3 and 4 were as good as Rose
Bengal in producing high levels of singlet oxygen. ROS production
measured by flow cytometric analysis of the fluorescein fluorescence
following treatment with equitoxic concentration of the BODIPYs,
corresponding to their IC50 values reported in Table 4, indicate that 3
and 4 induced a significant increase in ROS levels over control in
SKOV3 cells; specifically, in this cell line 3 was more effective than 4 in
generating ROS (Fig. 6). On the contrary, no significant increase in ROS
production was observed in HCT116 and MCF7 cells or when flow
cytometry was performed 24 h later (data not shown), indicating that
both ROS generation and persistence abate within 24 h from the end of
irradiation. These results suggest that 3 and 4 exert their photodynamic
activity on HCT116 and MCF7 cells preferentially through type II re-
action (singlet oxygen production) while type I and type II (ROS pro-
duction) reactions occurred simultaneously in SKOV3 cells.
Fig. 5. Uptake of 3 and 4 in HCT116, SKOV3 and MCF7 cells following 24 h
exposition to the BODIPYs (10 μM) (mean S.E. of 3–5 independent ex-
periments; ***p < 0.001 vs 4 same cell line).
±
Tumor cells of epithelial origin may possess intrinsic migratory
behavior, that has been reported as a mark of invasion and metastasis,
indicating that the cells have undergone an epithelial-to-mesenchimal
matter of fact their higher motility correlates with a higher degree of
malignancy, as compared to HCT116 and MCF7 cells.
The results showed in Fig. 7 indicate that, following photoactiva-
tion, subtoxic concentrations of 3 and 4 were able to slow down the
intrinsic migratory capacity of SKOV3 cells, albeit to varying degrees; in
the absence of irradiation none of the compounds were able to interfere
with the intrinsic migratory activity of SKOV3 cells (data not shown).
Analysis of the percentage of open scratch, performed using the
3
8,42
transition.
A number of PSs, such as a Photofrin derivative, have
38,43,44
shown anti-migratory activity.
However, no data concerning the
anti-migratory activity of BODIPY have been reported to date, thus, we
decided to evaluate the effect of 3 and 4 on cellular migration using the
scratch wound healing assay. Between the cell lines included in this
study, only SKOV3 cells were able to spontaneously migrate and as a
Fig. 4. Percentage of apoptotic (A) and
necrotic (B) HCT116, SKOV3 and MCF7
cells following PDT with equitoxic
concentrations of
3
and 4, corre-
sponding
to
the
IC50
values
(
mean ± S.E. of 3–5 independent ex-
periments). **p
< 0.01 vs control
same cell line; ***p < 0.001 vs control
same cell line; °p < 0.001 vs control
and 3 same cell line; #p < 0.001 vs
control and 4 same cell line.
5

E. Caruso, et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115737
Table 5
Percentage of open scratch wound in SKOV3 cells following 24 h treatment with
3
(0.75 nM) and 4 (2.5 nM), 2 h irradiation under green LED light and 24 h
incubation in drug-free medium at 37 °C. Pictures of the scratch wound were
taken immediately following the irradiation step (0) and after 24 h; percentage
of open scratch wound were evaluated by the TScratch software
(
mean ± S.E.M. of 3 independent experiments).
0
24 h
Control
38.27 ± 3.02
35.19 ± 2.61
37.71 ± 0.78
8.68 ± 0.58a
24.81 ± 2.54
13.53 ± 0.14
b
c
3
4
Fig. 6. ROS generation in HCT116, SKOV3 and MCF7 cells following 24 h ex-
posure or not (control) to 3 and 4 at their respective IC50 values and 2 h irra-
diation in compounds-free PBS. The intracellular ROS levels were determined
by flow cytometry at the end of the irradiation period. Mean ± S.E.M. of three
independent experiments. *** p < 0.001 vs controls and p < 0.05 vs 4, **
p < 0.001 vs controls.
a
b
c
p < 0.001 vs 0 same treatment.
p < 0.05 vs 0 same treatment and p < 0.01 vs control and 4 24 h.
p < 0.001 vs 0 same treatment and p < 0.05 vs control 24 h.
activity.
Furthermore, as supported by previously reported data, the pre-
sence of the bromine atom at the end of the alkyl chain could offer a
great synthetic ductility, allowing subsequent nucleophilic substitution
reactions either to introduce positive charges or to hook, through a
covalent bond, the BODIPY on nanoparticles for a more efficient drug
TScratch software, indicate that the antimigratory effect of 3 was sig-
nificantly higher than 4 (Table 5).
2
1
3
. Conclusions
delivery.
Apoptotic and necrotic cell deaths appear to play a pivotal role in
tumor cell death induced by the two BODIPYs, mediated by a singlet
oxygen and ROS level increase. Further investigations are required to
clearly explain the molecular mechanisms underlying the better re-
sponse observed with 3, compared with 4. However, the present data
suggest the potential use of 3 for PDT, also considering the interesting
results concerning the anti-migratory effect of this BODIPY.
Taken together, these data indicate that the presence of an appendix
consisting of linear carbon atoms chain of different length on the phenyl
group placed on the position 8 of the dipyrrolylmethene basic structure
give the two BODIPYs a different degree of lipophilicity, resulting in a
significant more efficient phototoxic effect of the molecule with lower
lipophilicity (i.e. 3). Thus, due to its high lipophilicity, compound 4
could probably form aggregates in the aqueous medium used for pho-
todynamic experiments, leading to a less effective photodynamic
A potential problem concerning the application of these BODIPYs
Fig. 7. Migratory activity of SKOV3 cells following
treatment with 3 (0.75 nM) and 4 (2.5 nM)for 24 h,
scratch formation, 2 h irradiation and incubation for
2
4 h in drug-free medium at 37 °C. Pictures of the
scratch wound were taken immediately following
the irradiation step (0) and after 24 h, through a
camera connected to an Olympus IX81 microscope.
6

E. Caruso, et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115737
for PDT, especially in tumors which are not superficial, is that ab-
sorption in the red region of the visible spectrum is required for clinical
applications, due to the ability of red light to penetrate deeper into the
tumor, whereas their optimal absorption wavelength lies in the green
region. However, the activation by green light can be useful when su-
perficial treatment is required and more penetrating wavelengths could
2H, CH
CH
2
); 2.13 (m, 2H, CH
2
); 2.58 (s, 6H, 2 × CH ); 3.55 (t, 2H,
3
2
2
Br); 4.08 (t, 2H, CH
2
O); 6.00 (s, 2H, 2 × CH ); 7.02 (d, 2H,
2 × CH); 7.19 (d, 2H, 2 × CH); HPLC retention time: 10′34′’ (100%).
The corresponding 2,6-diiodo compound (3) was obtained by
adding 431 mg of I
2
(1.545 mmol) and 299 mg of HIO (1.545 mmol) to
3
an ethanol solution of 3a (317 mg, 0.668 mmol). This mixture was
4
5
present a threat to contiguous organs. In addition, it has been re-
ported that a red-shift in the λmax could be easily gained by introducing
two styryl substituents in the α position of the BODIPY, thus obtaining
compounds featuring optimal absorption at approximately 650 nm,
which makes those particularly attractive in view of their possible use
stirred for 24 h at RT, washed with water and extracted three times with
CH
2
Cl
2
; the organic phase was dried over Na
2
4
SO and evaporated to
dryness. The crude product was purified by column chromatography
(SiO , CH Cl /petroleum ether = 1/1) affording 266 mg (0.37 mmol;
2
2
2
yield: 54.80%) of 14 as red needles.
4
6
as PSs for photodynamic cancer therapies. Finally, the emergence of
new technologies for photodynamic activation and light or PS delivery
is going to redefine the traditional views that PDT is limited to super-
Compound 3: C23
H
24BBrF
1
2
I
2
N
2
O M.W.: 726.97 g/mol; UV–Vis
) δ: 1.47 (s, 6H, 2 × CH );
); 2.62 (s, 6H, 2 × CH ); 3.54 (t,
(DCM): 533 nm (ε = 66072); H NMR (CDCl
3
3
2.04 (m, 2H, CH
2
); 2.16 (m, 2H, CH
2
3
4
7
ficial tumors. Moreover, in the long term, the availability of radio-
sensitizers, biocompatible nanoparticles, upconverting nanomaterials
and the ability to design personalized light delivery protocols could
make PDT a viable therapy that has significant impact in deep tissues
and enhances overall outcomes.
2H, CH
2
Br); 4.09 (t, 2H, CH
2
O); 7.04 (d, 2H, 2 × CH); 7.15 (d, 2H,
+
2 × CH); MS (ESI): M−1 found: 726.01 calcd for C23
2
H24BBrF I
2
N O
2
726.97; HPLC retention time: 14′16′’ (100%).
4.1.2. Synthesis of 2,6-diiodo-1,3,5,7-tetramethyl-8-{4-[(8-bromooctyl)
oxy]phenyl}-4,4′-difluoroboradiazaindacene (4)
4
. Materials and methods
1,3,5,7-tetramethyl-8-{4-[(8-bromooctyl)oxy]phenyl}-4,4′-di-
fluoroboradiazaindacene (4a) and 2,6-diiodo-1,3,5,7-tetramethyl-8-{4-
[(8-bromooctyl)oxy]phenyl}-4,4′-difluoroboradiazaindacene (4) were
4.1. Chemical
4
9
synthetized accordingly to the methods reported by Zagami et al.
UV–vis absorption spectra, 1H NMR spectra, mass spectrometric
measurements, HPLC analyses were performed as previously re-
4.1.3. Comparative Singlet-Oxygen generation measurements
4
8
1
ported. Merck 60 F254 silica gel precoated sheets (0.2 mm thick)
were used for analytical thin-layer chromatography (TLC). Silica gel 60
The ability of the two BODIPYs to generate
2
O was measured by
monitoring the disappearance of the 410 nm absorbance band of DPBF,
1
(
70–230 mesh, Merck) was used for column chromatographic separa-
a known O
2
scavenger and the relative singlet oxygen generation rates
tions. 4-hydroxybenzaldehyde, 1,4-dibromobutane, 1,8-dibromooctane
and 2,4-dimethylpyrrole were used as received by the supplier (Sigma-
for 3 and 4 were determined by comparison to the rate obtained with
Rose Bengal as previously reported.24
Aldrich). Dichloromethane (CH
2
Cl ), used in the synthesis of BODIPYs,
2
was freshly distilled directly into the reaction flask.
4.1.4. LogP determination
The green LED array is composed of 12 × 3 W diodes placed on a
The hydrophobicity was estimated using the software PaDEL-
50
2
7
28
1
1 cm diameter disk and equipped with a heat sinker. The emitted light
Descriptor and ACD Labs calculated LogP values i.e. Crippen LogP
28
is characterized by lambda max (λmax) of emission at 525 nm and width
and Galas LogP. Structural Description of the two BODIPYs (com-
pounds 3 and 4) and of their precursors (compounds 3a and 4a) was
encoded into SMILES strings which were used as input for the LogP
calculations. SMILES strings and calculated values are listed in Table 2.
−
3
2
at half maximum of 70 nm (fluence rate 3.036 × 10
W/cm ; light
2
energy density, or fluence, 21.8 J/cm ). A 50 W current transformer
provided electric supply. The LED array was placed above the samples
at such a distance as to produce a homogeneous area of irradiation.
4
.1.5. Photostability
4
.1.1. Synthesis of 4-[(4-bromobutyl)oxy]benzaldehyde (1) and of 4-[8-
The stability of BODIPYs 3 and 4 was evaluated using a UV-vis
bromooctyl)oxy]benzaldehyde (2)
spectrophotometer, by measuring every 20 min the decrease of the
absorbance profile of 10 µM solutions during 2 h irradiation with green
LED light. The decrease in the absorbance intensity al the lambda max
(530 nm) vs time plot was used to determine the photostability per-
centage.
4
-[(4-bromobutyl)oxy]benzaldehyde (1) and 4-[8-bromooctyl)oxy]
benzaldehyde (2) were synthetized accordingly to the methods reported
by Caruso et al.38
4
.1.1.1. Synthesis of 2,6-diiodo-1,3,5,7-tetramethyl-8-{4-[(4-bromobutyl)
oxy]phenyl}-4,4′-difluoroboradiazaindacene (3). 4-[(4-bromobutyl)oxy]
4.1.6. Fluorescence properties and quantum yield of fluorescence
The fluorescence properties of the BODIPYs were studied as pre-
viously reported.48
benzaldehyde and (838 mg, 3.2 mmol) and 2,4-dimethylpyrrole
(
724 μL, 7.04 mmol) were dissolved in dry CH
2
Cl
2
(60 mL) under N
2
atmosphere; then twenty drops of TFA were added and the solution was
stirred at RT overnight until the complete consumption of the aldehyde,
4.2. Biological
as determined by TLC analysis (SiO
2
,
CH
2
2
Cl ). Thereafter the
dipyrrolylmethane to dipyrrolylmethene oxidation was achieved by
4.2.1. Cell lines and photodynamic studies
adding solid DDQ (1.09 g, 4.8 mmol) and stirring the mixture for 2 h.
The human colon carcinoma HCT116 and the ovarian cancer SKOV3
cell lines were maintained in Dulbecco’s Modified Eagle Medium
(DMEM, Sigma-Aldrich), while the human breast cancer MCF7 cells
were cultured in Roswell Park Memorial Institute 1640 (RPMI1640,
Sigma-Aldrich) medium, supplemented with 10% fetal bovine serum,
1% glutamine, 0.25% neomycin and 0.5% penicillin–streptomycin
(Sigma-Aldrich). All the cell lines were maintained in standard culture
The last step of the BODIPY synthesis requires the addition of Et
3
N
.
(
5 mL) and BF
3
OEt (5 mL) to the reaction mixture. After 24 h under
2
stirring, the organic layer was washed two times with water, one time
with 1 M HCl solution and two more times with water; the organic
solution was then dried over Na
2
SO , filtered, and evaporated to
4
dryness. The raw material was purified by column chromatography
SiO , CH Cl /petroleum ether = 7/3) affording 317 mg (0.668 mmol,
yield: 20.9%) of the desired compound (3a) as orange needles.
26BBrF MW 475.18; UV–Vis (DCM): 503 nm
ε = 44100); H NMR (CDCl ) δ: 1.46 (s, 6H, 2 × CH ); 2.03 (m,
(
2
2
2
conditions at 37 °C in a humidified 5% CO atmosphere.
2
The effects of the BODIPYs on HCT116, SKOV3 and MCF7 cell
C
23
H
2
N
2
O
=
viability were evaluated by performing the MTT assay as previously
1
38,51,52
(
3
3
described.
Briefly, cells seeded in 96-well plates could attach for
7

E. Caruso, et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115737
2
4 h prior the exposition to the BODIPYs (0.1 and 200 nM) at 37 °C.
scratch wounds were evaluated statistically by analysis of the variance
After 24 h, the drug-containing medium was replaced by fresh PBS, and
cells were irradiated using a green LED source for 2 h. Cells were then
incubated in the dark at 37 °C in drug-free medium for 24 h and fol-
lowing 3 h incubation in the presence of MTT (2 mg/mL in PBS) ab-
sorbance of DMSO-dissolved formazan crystals was measured at
with Bonferroni post-test for multiple comparisons.
Funding
This study was supported by the Università degli Studi dell’Insubria,
Fondi di Ateneo per la Ricerca “FAR2018” and “FAR2019” (to MBG and
EC). The funding agencies were not involved in the research plan and
the interpretation of the data.
5
95 nm using Infinite® 200 PRO (Tecan). Possible intrinsic (i.e. not
photoinduced) effects of the BODIPYs were assessed by omitting the
irradiation step in cells treated with concentrations of BODIPY tenfold
higher (1–2000 nM) than those used for irradiated cells.
IC50 values were estimated from concentration-response curves by
non-linear regression analysis, using GraphPad Prism software, v. 5.0
Declaration of Competing Interest
(
GraphPad, San Diego, CA, USA).
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
4.2.2. Flow cytometric analysis
The ability of the tested BODIPY to induce apoptotic and/or necrotic
cell death and/or alterations in the distribution of DNA through the cell
cycle was evaluated, following 24 h exposition to 3 and 4 at their re-
spective IC50 (the values are reported in Table 4), 2 h irradiation in
drug-free PBS and 24 h incubation in drug-free medium in the dark, by
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